We have been investigating the regulation of Na+, K+-ATPase and associated Na+/K+-pump activities in synaptosomal preparations from rat brain. We have identified a novel substoichiometric mode of synaptosomal Na+, K+-ATPase (and associated Na+/K+-pump) inhibition by very low concentrations (IC50=10-15 M) of the specific steroid ligand ouabain. Substoichiometric inhibition by ouabain is transient and is observed under conditions that favor the Na+ and ATP-binding conformer (E1) of the enzyme. In addition, substoichiometric inhibition apparently involves a protein kinase A (PKA) and cyclooxygenase-dependent production oaa secondary Na+, K+-ATPase inhibitor that may be a cyclooxygenase-derived oxidant. Furthermore, ouabain structure-activities have led to the identification of the reduced lactone derivative of ouabain (dihydroouabain) as an antagonist of the substiochiometeric inhibition by ouabain. We have found that the low concentrations (IC20=5-10 mM) of ethanol also inhibit synaptosomal Na+, K+-ATPase and Na+/K+-pump activity via the PKA and cyclooxygenase-dependent mechanism. This novel mode of Na+, K+-ATPase inhibition by ethanol is evident under depolarizing conditions (i.e., veratridine) such that the depolarization-dependent activation of the synaptosomal Na+/K+-pump is inhibited by 10-25 mM ethanol. Importantly, the inhibition by ethanol was also found to be antagonized by dihydtoouabain. We are currently modifying the structure of dihydroouabain to promote its brain uptake so that the importance of Na+, K+-ATPase inhibition as a possible mediator of the central nervous system actions of ethanol can be delineated.